Contents and Abstracts of Latest Issue of BBB

(Vol.65 No.1 2001)

Soichi ARAI,・,a Toshihiko OSAWA,b Hajime OHIGASHI,c Masaaki YOSHIKAWA,d
Shuichi KAMINOGAWA,e Michiko WATANABE,f Tadashi OGAWA,d Kazuyoshi OKUBO,g
Shaw WATANABE,a Hoyoku NISHINO,h Kazuki SHINOHARA,i Takatoshi ESASHI,j
and Tsuneo HIRAHARA,k

aDepartment of Nutritional Science, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan
bNagoya University Graduate School of Bioagricultural Sciences, Chikusa-ku, Nagoya 464-8601, Japan
cGraduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
dResearch Institute for Food Science, Kyoto University, Uji-shi, Kyoto 611-0011, Japan
eGraduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku,
Tokyo 113-8657, Japan
fFaculty of Education, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan
gGraduate School of Agricultural Science, Tohoku University, Sendai-shi, Miyagi 981-8555, Japan
hDepartment of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan
iNational Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, Tsukuba,
Ibaraki 305-8642, Japan
jDepartment of Human Life and Culture, Seitoku University, Matsudo-shi, Chiba 271-8555, Japan
kCalpis Co. Ltd., Shibuya-ku, Tokyo 150-0021, Japan

The development of food science in the near future probably depends on the advance in functional food science, the concept of which was proposed first in Japan nearly 15 years ago. The new science has been internationally distributed and accepted as conceptually being beyond nutrition. In Japan, however, it traced a unique path of progress in the form of a product-driven rather than concept-driven science. Actually, a number of substances and products with potential for disease risk reduction rather than simply for health maintenance have been investigated for their body-modulating functions. Some of them have been applied in practice to the industrialization of functional foods in terms of ``foods for specified health uses′′ legally defined by new legislation. A variety of sophisticated methods have been introduced as well, including the so-called ``XYZ′′ evaluation system, database construction for assessment of the function, and even the DNA microarray technique. The Ministry of Agriculture, Forestry, and Fisheries (MAFF) and the Ministry of Health and Welfare (MHW) also commenced their scientific as well as political activity, with its spread to industries which almost simultaneously began to vigorously investigate functional food products for enlargement of the food market. With all of this as a background, the Japan Liaison of the International Union of Food Science and Technology (IUFoST) hold a function food science symposium on behalf of related scientific bodies including the Japan Section of the International Life Science Institute (ILSI). This paper is an overview compiled from 12 presentations made in the symposium, with the aim of internationally publicizing the activity of functional food science in Japan.
Key words: functional foods; food factors; foods for specified health uses; health claim

-2-
Detoxification Effect of Iron-encaging Zeolite-processed Water
in Tributyltin-intoxicated Euglena gracilis Z

Mari OHTA,1 Kaoru NAKAMURA,2 Tomokazu KUBO,1 and Tetsuya SUZUKI1,・

1Laboratory of Food Wholesomeness, Department of Marine Bioresources, Faculty of Fisheries,
Hokkaido University, 3-1-1 Minato, Hakodate 041-8611, Japan
2Institute of Chemical Research, Kyoto University, Gokasho, Uji 611-0011, Japan

Received March 17, 2000; Accepted September 19, 2000
In our previous paper, we reported the restoration promoting effects of mineral-encaging zeolite-processed water, especially of a Fe-encaging one, on tributyltin chloride (TBTCl)-intoxicated Euglena gracilis. This present study extends the investigation on the behavior of TBTCl and a xenobiotic enzyme, cytochrome P-450, in Euglena cells incubated with or without Fe-encaging zeolite-processed water (FeZW). Subcellular fractionation of TBTCl-intoxicated Euglena cells, atomic absorption spectrophotometry, and GC analyses showed that TBTCl was rapidly incorporated into the cells to halt cell motility. GC-MS showed that FeZW promoted conversion of TBTCl to dibutyltin (DBT) as the major metabolite in the microsomal fraction of the cells. An in vitro incubation system with heat-treated microsomes did not convert TBTCl to DBT. The contribution of cytochrome P-450 in the microsomal fraction was suggested by an immunochemical method. The results suggest that the improvement of detoxification by FeZW in the TBT-intoxicated Euglena cells should be due to activation of biotransformation system of the Euglena cells by FeZW.
Key words: tributyltin chloride; endocrine disrupter; Euglena; iron-encaging zeolite; detoxification

-3-
Molecular Cloning, Characterization, and Expression of Wheat Cystatins

Masaharu KURODA,＊ Toshihiro KIYOSAKI, Ichiro MATSUMOTO, Takumi MISAKA,
Soichi ARAI, and Keiko ABE・

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Received April 24, 2000; Accepted August 25, 2000
We cloned four kinds of cDNAs of wheat cystatins (WCs), WC1, WC2, WC3, and WC4, from the seed. They had 47--68％ amino acid sequence similarities to other plant cystatins. WC1, WC2, and WC4 had 63--67％ similalities to one another while 93％ of amino acids were identical between WC1 and WC3. This suggested that WC1, WC2, and WC4 should be regarded as the isoforms of wheat cystatins. The mRNAs for WC1, WC2, and WC4 were all expressed in seed at an early stage of maturation and, after that, their quantities decreased gradually. However, each of the mRNAs was again expressed one day after the start of germination and the expression continued for the following five days. WC1 seemed to be expressed at a higher level than WC2 and WC4. Immunostaining for looking at site-specific expression of each WC demonstrated that both WC1 and WC4 existed in the aleuron layer and embryo, but in the endosperm the only existing species was WC1. Differences in mRNA level and tissue localization found for the WCs may suggest their differential physiological roles.
Key words: cysteine proteinase inhibitor; cystatin; cysteine proteinase; Triticum aestivum; wheat seed

-4-
Improved Procedure for the Enantiometric Synthesis
of 1-Hydroxy/acetoxy-2,6-diaryl-3,7-dioxabicyclo[3.3.0]octane Lignans:
Total Syntheses of (+)-Paulownin, (+)-Phrymarin I and (+)-Phrymarin II

Fumito ISHIBASHI,1,・ Mami HAYASHITA,1 Momotoshi OKAZAKI,2 and Yoshihiro SHUTO2

1Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan
2Faculty of Agriculture, Ehime University, Matsuyama 790, Japan

Received May 1, 2000; Accepted August 2, 2000
Short enantiomeric syntheses of the 1-hydroxy/acetoxy-3,7-dioxabicyclo[3.3.0]octane lignans, (+)-paulownin, and (+)-phrymarin I and II, were accomplished by starting from the chiral synthon, (R)-(+)-3-hydroxybutanolide, and employing photocyclization as the key step.
Key words: enantiomeric synthesis; (+)-paulownin; (+)-phrymarin I; (+)-phrymarin II; photocyclization

-5-
New 11(15→1)Abeotaxane, 11(15→1),11(10→9)Bisabeotaxane
and 3,11-Cyclotaxanes from Taxus yunnanensis

Hiromasa KIYOTA,・,1 Qing-wen SHI,1,2 Takayuki ORITANI,1 and Ligeng LI2

1Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural
Science, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
2Department of Chemistry of Medicinal Natural Products, Faculty of Pharmaceutical Science,
Hebei Medical University, 361 Zhongshan East Street, Shijiazhuang 050017, P.R.China

Received May 1, 2000; Accepted August 2, 2000
Chemical examination of the seeds of Chinese yew, Taxus yunnanensis Cheng et L. K. Fu resulted in the isolation of an 11(15→1)abeotaxane, an 11(15→1),
11(10→9)bisabeotaxane and two 3,11-cyclotaxanes. The structures of these new taxoids were established as 13α-acetoxy-5α-cinnamoyloxy-11(15→1)abeotaxa-4(20),11-diene-9α,10β,15-triol (1), 20-acetoxy-2α-benzoyloxy-4α,5α,7β,9α,13α-pentahydroxy-11(15→1),11(10→9)
bisabeotax-11-eno-10,15-lactone (2), 2α,10β-diacetoxy-5α-cinnamoyloxy-9α-hydroxy-3,11-cyclotax-4(20)-en-13-one (3) and 10β-acetoxy-2α,5α,9α-trihydroxy-3,11-cyclotax-4(20)-en-13-one (4) on the basis of spectral analyses.
Key words: Taxus yunnanensis Cheng et L. K. Fu; taxanes; 11(15→1)abeotaxane; 11(15→1),11(10→9)bisabeotaxane; 3,11-cyclotaxane

-6-
Importance of the Carbohydrate-Binding Module of Clostridium stercorarium
Xyn10B to Xylan Hydrolysis

Mursheda K. ALI, Hidenori HAYASHI, Shuichi KARITA,＊ Masakazu GOTO,
Tetsuya KIMURA, Kazuo SAKKA,・ and Kunio OHMIYA

Faculty of Bioresources and ＊Center for Molecular Biology and Genetics, Mie University, Tsu 514-8507, Japan

Received May 12, 2000; Accepted August 21, 2000
The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596--1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BΔCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-
resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.
Key words: xylanase; Clostridium stercorarium; carbohydrate-binding module; cellulose-binding domain; xylan-binding domain

-7-
Volatile Flavor Components of Ripe and Overripe Ki-mikans
(Citrus flaviculpus Hort. ex Tanaka) in Comparison with Hyuganatsu
(Citrus tamurana Hort. ex Tanaka)

Hyang-Sook CHOI and Masayoshi SAWAMURA＊

Department of Bioresources Science, Faculty of Agriculture, Kochi University, B-200 Monobe,
Nankoku, Kochi 783-8502, Japan

Received May 31, 2000; Accepted August 3, 2000
The volatile flavor components of ripe and overripe ki-mikan (Citrus flaviculpus Hort. ex Tanaka) peel oil samples, which had been isolated by cold-pressing, were investigated by capillary GC and GC-MS, and compared with the Hyuganatsu (Citrus tamurana Hort. ex Tanaka) flavor. Limonene (ripe fruit, 82.44％; overripe fruit, 73.10％) was the most abundant compound in the ki-mikan oil, this being followed by γ-terpinene (8.83％ and 13.74％), trans-β-farnesene (1.76％ and 3.12％) and myrcene (1.54％ and 1.13％). The composition of overripe ki-mikan oil was characterized by higher amounts of aliphatic and sesquiterpene hydrocarbons, monoterpene and sesquiterpene alcohols, ketones and esters than that of ripe ki-mikan oil. Monoterpene hydrocarbons, especially limonene (84.78％), were predominant in Hyuganatsu oil. The CPO composition of ki-mikan was qualitatively similar to that of Hyuganatsu, but differed quantitatively. The content of sesquiterpene hydrocarbons was higher in the ki-mikan oil samples than in Hyuganatsu oil, while ketones showed the opposite predominance. These differences were more evident in the trans-β-farnesene and l-carvone contents. The ratio of both these compounds could be used to distinguish ki-mikan oil from Hyuganatsu oil.
Key words: ki-mikan (Citrus flaviculpus Hort. ex Tanaka); cold-pressed peel oil; ripening stage; volatile flavor components; trans-β-farnesene; l-carvone

-8-
Molecular Cloning of Rat USF2 cDNA and Characterization
of Splicing Variants

Kyoko TAKAHASHI,1 Chiharu NISHIYAMA,1,3 Ko OKUMURA,2,3 Chisei RA,2,3
Yasuyuki OHTAKE,1 and Toyokazu YOKOTA1

1Foods and Pharmaceuticals Research and Development Laboratory, Asahi Breweries Ltd., 1-1-21,
Midori, Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan
2Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku,
Tokyo 113-8421, Japan
3Allergy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku,
Tokyo 113-8421, Japan

Received June 9, 2000; Accepted August 18, 2000
The complete nucleotide sequence of rat USF2 cDNA was determined. In addition to the full length clone (USF2FL), four isoforms (Δ1, Δ2, Δ3, and Δ4) suggested to be generated by alternative splicing were isolated. USF2Δ1 and Δ2 lacked 27 and 67 internal amino acid residues, respectively. USF2Δ3 and Δ4 lacked most of the entire sequence but encoded short peptides of an N-terminal portion of USF2FL. Overexpression of USF2FL increased the transcription of the human high affinity IgE receptor (FcεRI) α chain gene through specific binding to the CAGCTG motif in the first intron. On the other hand, overexpression of USF2Δ1 or Δ2 reduced the transcription of the human FcεRI α chain gene. Both USF2FL and USF2Δ1 bound to CACGTG as well as CAGCTG, while USF2Δ2 bound to CACGTG but not to CAGCTG. These results suggested the presence of a different and definitive role of each variant in the expression of the α chain gene.
Key words: USF2; rat; splicing; isoform

-9-
Purification and Some Properties of Ubiquinol Oxidase from Obligately
Chemolithotrophic Iron-oxidizing Bacterium,
Thiobacillus ferrooxidans NASF-1

Kazuo KAMIMURA,＊ Shinji FUJII, and Tsuyoshi SUGIO

Department of Biological Function, Faculty of Agriculture, Okayama University,
1-1-1 Tsushima-Naka Okayama 700-8530, Japan

Received June 15, 2000; Accepted September 19, 2000
Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T. ferrooxidans. The ubiquinol oxidase was purified 79-fold from plasma membranes of T. ferrooxidans NASF-1 cells. The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d. Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively. This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm. These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear. The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N?,N?-tetramethyl-p-phenylenediamine dihydrochloride. The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E. coli.
Key words: Thiobacillus ferrooxidans; ubiquinol oxidase; iron-oxidizing bacterium; acidophile; cytochrome b

-10-
Soy Protein Hydrolyzate with Bound Phospholipids Reduces Serum
Cholesterol Levels in Hypercholesterolemic Adult Male Volunteers

Goro HORI,1 Ming-Fu WANG,2 Yin-Ching CHAN,2 Tatsuji KOMATSU,3 Yueching WONG,4
Tzu-Hsiu CHEN,3 Kazuhiro YAMAMOTO,1 Satoshi NAGAOKA,5 and Shigeru YAMAMOTO3

1Health Care Products Department, Kyowa Hakko Kogyo Co., Ltd., Tokyo 100-8185, Japan
2Department of Food and Nutrition, Provident University, Taichung, Taiwan
3Department of Nutrition, School of Medicine, Tokushima University, Tokushima 770-8503, Japan
4Department of Nutrition, Chungshan Medical and Dental Collage, Taichung, Taiwan
5Department of Food Science, Faculty of Agriculture, Gifu University, Gifu 501-1112, Japan

Received June 19, 2000; Accepted August 26, 2000
This study was done to evaluate the effects of soy protein hydrolyzate with bound phospholipids (c-SPHP), on the serum cholesterol levels in hypercholesterolemic subjects over a three-month period. Subjects were Taiwanese adult male volunteers whose serum total cholesterol levels were above 220 mg/dl. Twenty-one subjects were divided into three groups randomly, and each group was given c-SPHP zero, 3, or 6 g per day. Test diets were orally administered in a powdered drink form that contained c-SPHP or casein hydrolyzate (placebo). The subjects were given the test diet four times daily. The study consisted of a two-week pre-feeding period, a three-month feeding period, followed by a two-week post-feeding period. After 3 months of c-SPHP administration, 3 g per day, serum total cholesterol decreased significantly from the initial level (15.0％, p<0.01) and compared with the placebo group (p<0.05). Furthermore, LDL-cholesterol decreased significantly (27.7％, p<0.01) and the LDL/HDL ratio also decreased significantly (47.4％, p<0.01) from the initial levels. These effects of c-SPHP were dose-dependent. This study suggests that c-SPHP has remarkable improving effects on the serum cholesterol levels in hypercholesterolemic subjects.
Key words: soy protein; phospholipid; serum cholesterol; humans; hypercholesterolemia

-11-
Direct Expression of the Extracellular Portion of Human FcεRIα Chain
as Inclusion Bodies in Escherichia coli

Toshiro TAKAI,1,2,・ Ko OKUMURA,1 and Chisei RA1,3

1Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku,
Tokyo 113-8421, Japan
2Bioscience Research and Development Laboratory, Asahi Breweries, Ltd., 1-21 Midori 1-chome,
Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan
3Division of Molecular Biology, Allergy Research Center, Juntendo University School of Medicine,
2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

Received June 23, 2000; Accepted August 10, 2000
The extracellular portion of the α chain of the human high-affinity IgE receptor (FcεRIα) was expressed as inclusion bodies in Escherichia coli. In immunoblot analysis, two bands were reactive to human IgE and mouse anti-human FcεRIα monoclonal antibodies. N-terminal sequencing showed that the two bands were equivalent to the soluble FcεRIα with a methionine residue at the N-terminus (Met-1-172) and 23-172, in which the N-
terminal 22 residues of the soluble FcεRIα have been removed, possibly by degradation in E. coli cells. IgE-binding to CHO cells expressing FcεRI was inhibited by the addition of the recombinant products prepared by the refolding procedure from inclusion bodies. The system for the expression of soluble human FcεRIα in E. coli presented in this study and its further improvement would be useful for the production of the protein as a potent therapeutic and for analysis of the IgE-FcεRIα interaction.
Key words: allergy; high-affinity IgE receptor; Escherichia coli; expression system; inclusion body

-12-
Domain Construction of Cherry-tomato Lectin: Relation
to Newly Found 42-kDa Protein

Yoshikazu NAITO, Tomoyuki MINAMIHARA, Akikazu ANDO, Tazunu MARUTANI,
Suguru OGURI,・ and Yoshiho NAGATA・・

Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University, Matsudo 271-8510, Japan

Received June 27, 2000; Accepted September 5, 2000
In the early stage of ripening of cherry-tomato fruits (Lycopersicon esculentum var. cherry), the lectin activity increased logarithmically and reached a plateau at day 10 after flowering. During purification of lectin from ripe and unripe fruits, a 42-kDa protein was found abundantly in unripe fruits. The protein cross-reacted with anti-cherry-tomato-lectin serum, retained chitin-binding ability, but showed no lectin activity. Comparative studies between the structure of the lectin and the 42-kDa protein were done. N-Terminal amino acid sequences of the lectin, peptides derived from the S-pyridylethylated lectin, and fragments generated by limited proteolysis of the native lectin showed that the lectin was comprised of three domains, Hyp-rich, Cys-rich, and Gln-rich, and the alignment of them was as this order from the N-terminus. Studies on the 42-kDa protein showed that it contained two of the three domains, Cys-rich and Gln-rich, but the amino acid sequence analysis showed that the protein should be a product of another gene.
Key words: Cys-rich chitin-binding domain; Hyp-rich glycoprotein domain; Lectin-related 42-kDa protein; Lycopersicon esculentum var. cherry; Structure of cherry-tomato lectin

-13-
High-Level Secretory Production of Phospholipase A1 by Saccharomyces
cerevisiae and Aspergillus oryzae

Yoichiro SHIBA,＊,・ Chiho ONO,＊ Fumio FUKUI,＊ Ichiro WATANABE,＊＊ Nobufusa SERIZAWA,＊＊
Katsuya GOMI,＊＊＊ and Hiroji YOSHIKAWA＊

＊Lead Discovery Research Laboratories, Sankyo Co. Ltd., 389-4 Aza-Ohtsurugi, Shimokawa, Izumimachi,
Iwaki, Fukushima 971-8183, Japan
＊＊Biomedical Research Laboratories, Sankyo Co. Ltd., 1-2-58, Hiromachi, Shinagawa-ku,
Tokyo 140-8710, Japan
＊＊＊Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University,
Sendai, Miyagi 981-8555, Japan

Received June 30, 2000; Accepted September 14, 2000
Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of lecithin to form lysolecithin. The PLA1 gene, which had been cloned from Aspergillus oryzae, was expressed in Saccharomyces cerevisiae and A. oryzae. Through the modification of the medium composition and the feeding conditions of substrate, the production level of PLA1 by S. cerevisiae was increased to a level fivefold higher than that indicated in a previous report. In the case of A. oryzae, introduction of multicopies of PLA1 expression units, and the morphological change from the pellet form to the filamentous form were effective for the enhancement of PLA1 production. We succeeded in producing 3,500 U/ml of PLA1 using an industrial-scale fermentor.
Key words: phospholipase A1; Saccharomyces cerevisiae; CPY; Aspergillus oryzae; mycelial morphology

-14-
Purification and Properties of Thiosulfate Dehydrogenase
from Acidithiobacillus thiooxidans JCM7814

Kazuo NAKAMURA, Maya NAKAMURA, Hiroshi YOSHIKAWA, and Yoshifumi AMANO

Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University,
Kofu 400-8511, Japan

Received July 3, 2000; Accepted September 26, 2000
A key enzyme of the thiosulfate oxidation pathway in Acidithiobacillus thiooxidans JCM7814 was investigated. As a result of assaying the enzymatic activities of thiosulfate dehydrogenase, rhodanese, and thiosulfate reductase at 5.5 of intracellular pH, the activity of thiosulfate dehydrogenase was measured as the key enzyme. The thiosulfate dehydrogenase of A. thiooxidans JCM7814 was purified using three chromatographies. The purified sample was electrophoretically homogeneous. The molecular mass of the enzyme was 27.9 kDa and it was a monomer. This enzyme had cytochrome c. The optimum pH and temperature of this enzyme were 3.5 and 35°C. The enzyme was stable in the pH range from 5 to 7, and it was stable up to 45°C. The isoelectric point of the enzyme was 8.9. This enzyme reacted with thiosulfate as a substrate. The Km was 0.81 mM.
Key words: thiosulfate dehydrogenase; Acidithiobacillus thiooxidans

-15-
Isolation of Antidiabetic Components from White-Skinned Sweet Potato (Ipomoea batatas L.)

Shuichi KUSANO, Hiroyuki ABE, and Hirohide TAMURA

Research Institute, Fuji Sangyo Co., Ltd., Tamura-cho 1301, Marugame, Kagawa 763-0071, Japan

Received July 3, 2000; Accepted August 28, 2000
We have already reported that white-skinned sweet potato (Ipomoea batatas L.) (WSSP) shows antidiabetic activity in streptozotocin (STZ) induced diabetic rats and genetically diabetic models (yellow KK, db/db mice and Zucker fatty rats). In this study, isolation and purification of the antidiabetic component of WSSP were attempted. Almost all antidiabetic activity was found in the cortex of WSSP. The fractionation of the antidiabetic component in the WSSP cortex was done by the following methods: dialysis of the water extract, 85％ ethanol precipitation, 15％ trichloroacetic acid (TCA) treatment, butyl-, phenyl-hydrophobic column chromatography, and ultrafiltration treatment. The antidiabetic component was not eliminated during dialysis and was soluble in 85％ ethanol and 15％ TCA, but it passed through a filter that allows the passage of substances of a molescular weight of 30,000. The uniformity of this isolated active component was analyzed using HPLC. A single peak was seen with three different columns (C8 reverse-phase column, anion exchange QA column, and gel filtration column (GFC)), indicating that the component is a uniform substance. The molecular weight of this antidiabetic component was estimated to be 22,000 by GFC analysis. This active component was presumed to be an acidic glycoprotein because it contained protein and sugar and was adsorbed onto the QA column at pH 7.0.
Key words: white skinned sweet potato; antidiabetic component; normal rats; hypoglycemic; db/db mice

-16-
Membrane-bound Sugar Alcohol Dehydrogenase in Acetic Acid Bacteria
catalyzes L-Ribulose Formation and NAD-Dependent Ribitol Dehydrogenase is
Independent of the Oxidative Fermentation・・・

Osao ADACHI,・ Yoshikazu FUJII, Yoshitaka ANO, Duangtip MOONMANGMEE,・・
Hirohide TOYAMA, Emiko SHINAGAWA,＊ Gunjana THEERAGOOL,＊＊ Napha LOTONG,＊＊
and Kazunobu MATSUSHITA

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
＊Department of Chemical and Biological Engineering, Ube National College of Technology, Tokiwadai,
Ube 755-8555, Japan
＊＊Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand

Received July 10, 2000; Accepted September 13, 2000
To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100 kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5--10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be the enzyme responsible for L-ribulose production in oxidative fermentation by acetic acid bacteria.
Key words: acetic acid bacteria; Gluconobacter suboxydans; oxidative fermentation; pentitol oxidation; ribitol dehydrogenase

-17-
Effects of pH and Metal Ions on Antioxidative Activities of Catechins

Midori KUMAMOTO,・ Tamiyoshi SONDA, Kinuyo NAGAYAMA, and Masaaki TABATA＊

Department of Food and Nutrition, Faculty of Home Science, Nishikyushu University, Kanzaki-machi,
Saga 842-8585, Japan
＊Department of Chemistry, Faculty of Science and Engineering, Saga University, Honjyo-machi,
Saga 840-8502, Japan

Received July 14, 2000; Accepted September 1, 2000
The Effects of pH on antioxidative activities of catechol, pyrogallol, and four catechins, and effects of metal ions (Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, and Zn2+) on antioxidative activities of (--)-epigallocatechin gallate (EGCG) were studied by an oxygen electrode method. The antioxidative activities of catechins were high and constant at pH 6--12, but decreased in acidic and strong alkaline solutions. Copper(II) ion the most strongly increased the antioxidative activity of EGCG among these metal ions examined, but iron(II) ion largely inhibited the antioxidative activity of EGCG. These effects are discussed considering the formation of metal complexes with catechins and the change in oxidation potentials.
Key words: antioxidative activity; catechins; metal ions; oxidation potential

-18-
Purification and Characterization of Bifunctional Alginate Lyase
from Alteromonas sp. Strain No. 272 and Its Action
on Saturated Oligomeric Substrates

Yoshiko IWAMOTO,1 Ryoko ARAKI,1 Ken-ichi IRIYAMA,1 Tatsuya ODA,1 Hisataka FUKUDA,2
Shinziro HAYASHIDA,2 and Tsuyoshi MURAMATSU1,・

1Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan
2Research and Development Division, Choko Shoyu Miso Co-op., Mizoriku-machi, Omura,
Nagasaki 856-0844, Japan

Received July 17, 2000; Accepted September 11, 2000
A marine bacterium (strain No. 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species. The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS. The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8. The predominant secondary structure of the enzyme was found to be most likely β-structure by circular dichroism. The enzyme was most active at pH 7.5--8.0 and stable around pH 5--11. The enzyme was more labile in Tris-HCl buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0). No of metal ions significantly affected the enzyme activity. The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-α1,4-L-guluronate and poly-β1,4-D-mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates. However, the coexisting poly-α1,4-L-guluronate and poly-β1,4-D-mannuronate apparently interacted with the enzyme in a competitive manner. Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and mannuronate, but on the tetramers or more. The kinetic analyses showed that kcat/Km for each oligomer was larger for the guluronate oligomers than for the mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (kint) and intrinsic substrate binding constant (Kint).
Alteromonas sp.; bifunctional alginate lyase; guluronate; mannuronate; subsite structure

-19-
Development of Assay System for Immunoglobulin Production Regulatory
Factors using Whole Cell Cultures of Mouse Splenocytes

Mikako TAKASUGI, Yuki TAMURA,＊ Hirofumi TACHIBANA,＊
Michihiro SUGANO,＊＊ and Koji YAMADA＊

Faculty of Human Life Science, Yamaguchi Prefectural University, 3-2-1 Sakurabatake,
Yamaguchi 753-8502, Japan
＊Division of Bioresource and Bioenvironmental Sciences, Graduate School of Kyushu University,
6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
＊＊Faculty of Environmental and Symbiotic Science, Prefectural University of Kumamoto, 3-1-100 Tsukide,
Kumamoto 862-8502, Japan

Received July 17, 2000; Accepted August 29, 2000
We tried to establish an assay system for screening and assessment of immunoregulatory factors using whole cell cultures of mouse splenocytes and found that splenic adhesive cells markedly increased immunogobulin (Ig) production of splenocytes. In the absence of adhesive cells, lipopolysaccharides, pokeweed mitogen, and phytohemagglutinin stimulated the production of IgA, IgG, and IgM in a class-dependent manner. Adhesive cells increased more markedly Ig production of splenocytes stimulated with these mitogens. When mouse splenocytes were cultured with milk proteins in the absence of adhesive cells, lactoferrin, β-lactoglobulin, α-casein, and β-casein stimulated IgA and IgG production. Adhesive cells increased IgA production of splenocytes stimulated with milk proteins, especially. These results suggest that the assay system is useful for assessment of Ig production-regulating factors.
Key words: adhesive cells; milk proteins; mitogens; immnoglobulin; splenocytes

-20-
Cloning and Analysis of Valerophenone Synthase Gene Expressed Specifically
in Lupulin Gland of Hop (Humulus lupulus L.)

Yukio OKADA and Kazutoshi ITO

Plant Bioengineering Research Laboratories, Sapporo Breweries Ltd., 37-1, Kizaki, Nitta,
Gumma 370-0393 Japan

Received July 28, 2000; Accepted August 24, 2000
Resin and essential oil derived from hop (Humulus lupulus L.) cones are very important compounds for beer brewing, and they specifically accumulate in the lupulin gland of hop cones. In order to identify the genes responsible for the biosynthetic pathway of these compounds and use the identified genes for hop breeding using Marker Assisted Selection and transformation techniques, genes expressed specifically in the lupulin gland were cloned and sequenced. One of them was suggested to be similar to the chalcone synthase gene from the DNA sequence. The translation product of the gene had the activity of valerophenone synthase, which catalyzes a part of the synthesis reaction of α-acid and β-acid. Northern analysis showed that the valerophenone synthase gene seemed to be expressed specifically in the lupulin gland.
Key words: hop; Humulus lupulus; valerophenone synthase; chalcone synthase; α-acid

-21-
Note
A Calcium-binding Protein with Four EF-hand Motifs
in Streptomyces ambofaciens

Tohru YONEKAWA, Yasuo OHNISHI, and Sueharu HORINOUCHI・

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan

Received April 17, 2000; Accepted August 25, 2000
A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.
Key words: calmodulin-like protein; EF-hand; calcium-binding protein; Streptomyces ambofaciens

-22-
Note
Syntheses and Biological Activities of Pyranyl-substituted Cinnamates

Jun ZHU, Motoji MAJIKINA, and Shinkichi TAWATA・

Faculty of Agriculture, University of the Ryukyus, Senbaru 1, Nishihara-cho, Nakagami-gun,
Okinawa 903-0129, Japan

Received April 27, 2000; Accepted August 18, 2000
Twenty-two kinds of pyranyl-substituted cinnamates were synthesized by the reaction of 4-hydroxy-6-(2-phenylethyl)-2H-pyran-2-one or 4-hydroxy-6-methyl-2H-pyran-2-one (HMP) with a variety of substituted cinnamic acids, and their antifungal and plant growth inhibitory activities were investigated. Among the compounds prepared, 6-methyl-2-oxo-2H-pyran-4-yl 3-(4-isopropylphenyl)propenoate (H5) showed the strongest antifungal activity against Rhizoctonia solani and Sclerotium dellfinii, and 6-methyl-2-oxo-2H-pyran-4-yl 3-(2-methylphenyl)propenoate (H2) had the highest plant growth inhibitory activity toward Brassica rapa.
Key words: cinnamate; pyran; 7,8-dihydro-5,6-|||dehydrokawain; antifungal activity; plant growth inhibition

-23-
Note
Intraspecific Divergence of Saccharomyces kluyveri as Revealed
by the Nucleotide Sequences of 18S-28S rRNA Spacer Regions
and α-Galactosidase MEL Genes

Yuji ODA and Tomoko FUJISAWA＊

Upland Agriculture Research Center, Hokkaido National Agricultural Experiment Station, Memuro,
Kasai, Hokkaido 082-0071, Japan
＊Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan

Received May 17, 2000; Accepted August 22, 2000
In the five strains classified as the yeast Saccharomyces kluyveri, several substitutions were observed in the two internal transcribed spacer regions between 18S and 28S rRNA. A PCR reaction with primers targeted to the MEL1 gene of Saccharomyces cerevisiae amplified fragments of the expected size, and those sequences showed significant divergence in the strains of S. kluyveri.
Key words: Saccharomyces kluyveri; ITS ribosomal RNA; α-galactosidase; MEL

-24-
Note
Synthesis of the Racemate of the Stereoisomer at C-6a of BE-40644,
a Bioactive Metabolite of Actinoplanes sp. with a Sesquiterpene-substituted
p-Benzoquinone Structure・

Hisayuki TSUJIMORI・・ and Kenji MORI・・・

Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku,
Tokyo 162-8601, Japan

Received May 29, 2000; Accepted August 14, 2000
BE-40644 is a tetracyclic metabolite of Actinoplanes sp. A 40644 possessing a sesquiterpene-substituted p-benzoquinone structure with cis-fused B/C ring stereochemistry that inhibits the human thioredoxin system as the well as the growth of several cancer cell lines. Its B/C trans-fused stereoisomer at C-6a was synthesized as a racemate starting from geranylacetone and 3,5-dihydroxybenzoic acid.
Key words: Actinoplanes sp.; heterocycle; microbial metabolite; quinone; terpenoid

-25-
Note
Synthesis of Corollosporine, an Antibacterial Metabolite
of the Marine Fungus Corollospora maritima・

Tomoya OHZEKI and Kenji MORI・・

Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3,
Shinjuku-ku, Tokyo 162-8601, Japan

Received June 5, 2000; Accepted August 2, 2000
Corollosporine [(±)-3-hexyl-3,7-dihydroxy-1(3H)-isobenzofuran-1-one], an antibacterial metabolite of the marine fungus, Corollospora maritima, was synthesized by four different routes from 3-hydroxyphthalic anhydride or 2-methoxybenzoic acid as the starting material to verify its proposed structure.
Key words: antibacterial metabolite; Corollospora maritima; lactone; marine fungus; phthalide

-26-
Note
Monomolecular Layer Formation of Ferritin Molecules on an Amphiphilic
Cyclodextrin Derivative at the Air/Water Interface

Kazutoshi KOBAYASHI,＊,＃ Noriyuki ISHII,＊＊,・ Hiroyuki SASABE,＊,＃＃
and Wolfgang KNOLL＊,＊＊＊,＃＃＃

＊Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan
＊＊Biophysical Chemistry Laboratory, National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology, Ministry of International Trade and Industry,
1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
＊＊＊Max-Planck-Institut f"ur Polymerforschung, Ackermannweg 10, D-55128 Mainz, Germany

Received June 13, 2000; Accepted August 22, 2000
A monomolecular layer of ferritin molecules was formed by adsorption from the subphase onto a Langmuir film of an amphiphilic β-cyclodextrin (β-CD) derivative at the air/water interface. The course of the adsorption of ferritin molecules was monitored by measuring the surface pressure and the resulting film was observed by transmission electron microscopy (TEM). These results show the potential of the amphiphilic CD derivative to work as a milder template for protein molecules at the air/water interface.
Key words: monolayer; ferritin; cyclodextrin derivative; surface pressure; transmission electron microscopy

-27-
Note
An Efficient Synthesis of C2-Symmetric Chiral Binaphthyl Ketone Catalysts

Toshiyuki FURUTANI, Masanori HATSUDA, Toshiaki SHIMIZU, and Masahiko SEKI＊

Product ＆ Technology Development Laboratory, Tanabe Seiyaku Co. Ltd., 3-16-89 Kashima, Yodogawa-ku, Osaka 532-8505, Japan

Received June 21, 2000; Accepted August 21, 2000
An efficient synthesis of C2-symmetric chiral binaphthyl ketones 1a and b, effective catalysts for asymmetric epoxidation, is reported. The key features of this synthesis are Co(salen)-catalyzed macrolactonization of racemic 1,1?-binaphthyl-2,2?-dicarboxylic acid monoglycidyl esters 3a and b and lipase-catalyzed enantioselective acylation of resulting 11-membered cyclic binaphthyl alcohols 4a and b.
Key words: biaryls; enzymic reaction; kinetic resolution; acylation

-28-
Note
A Simple, Rapid, and Highly Efficient Gene Expression System
for Multiheme Cytochromes c

Kiyoshi OZAWA,1 Fumiko YASUKAWA,1 Yumiko FUJIWARA,1 and Hideo AKUTSU1,2,・

1Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University,
Hodogaya-ku, Yokohama 240-8501, Japan
2Institute for Protein Research, Osaka University, Yamadaoka, Suita 565-0871, Japan

Received June 23, 2000; Accepted August 23, 2000
The genes of tetraheme cytochrome c3 and hexadecaheme high-molecular-weight cytochrome c from Desulfovibrio vulgaris could be overexpressed as holoproteins in Shewanella oneidensis TSP-C using pUC-type vectors of E. coli. Surprisingly, S. oneidensis was transformed directly by pUC-type vectors through electroporation. The yields of the recombinant proteins in this expression system were much higher than the previously reported ones.
Key words: multiheme cytochrome c; Shewanella oneidensis; pUC-type vector; electroporation

-29-
Note
Isolation of the rpoD Gene Encoding the Principal Sigma Factor
of the Deep-sea Piezophilic Bacterium Shewanella violacea Strain DSS12
and Its Overexpression in Escherichia coli

Kaoru NAKASONE,＊,・ Akihiko IKEGAMI,＊,＊＊ Chiaki KATO,＊ and Koki HORIKOSHI＊,＊＊

＊The DEEP STAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho,
Yokosuka, Kanagawa 237-0061, Japan
＊＊Department of Applied Chemistry, Faculty of Engineering, Toyo University, 2100 Kujirai, Kawagoe,
Saitama 350-0852, Japan

Received June 28, 2000; Accepted August 18, 2000
The gene encoding the principal σ factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced. The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3％ identity to those of Escherichia coli and Pseudomonas putida, respectively. Comparison with E. coli σ70 and P. putida σ70 showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding. Using an expression plasmid harboring the rpoD gene, the S. violacea σ70 factor was overexpressed in E. coli and successfully purified to near homogeneity.
Key words: deep-sea piezophilic bacterium; Shewanella violacea; principal sigma factor; rpoD; transcription

-30-
Note
In Vitro Absorption and Metabolism of Nobiletin, a Chemopreventive
Polymethoxyflavonoid in Citrus Fruits

Akira MURAKAMI,1 Shigeru KUWAHARA,2 Yasuo TAKAHASHI,2 Chihiro ITO,3
Hiroshi FURUKAWA,3 Motoharu JU-ICHI,4 Koichi KOSHIMIZU,1
and Hajime OHIGASHI5,・

1Department of Biotechnological Science, Faculty of Biology-oriented Science and Technology,
Kinki University, Iwade-Uchita, Wakayama 649-6493, Japan
2Research and Development Division, Wakayama Agricultural Processing Research Corporation,
398 Tsukatsuki Momoyama-cho, Nagagun, Wakayama 649-6112, Japan
3Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan
4Faculty of Pharmaceutical Sciences, Mukogawa Women′s University, Nishinomiya, Hyogo 663-8179, Japan
5Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received June 30, 2000; Accepted August 10, 2000
The polymethoxyflavonoid (PMF), nobiletin (NOB), specifically occurs in citrus fruits, and is currently believed to be a promising anti-inflammatory and anti-tumor promoting agent. In the present study, we investigated the in vitro absorption and metabolism of NOB and compared them with those of the polyhydroxyflavonoid (PHF), luteolin (LT). NOB preferentially accumulated in a differentiated Caco-2 cell monolayer, which is a model for small intestinal epithelial cells, while LT did not. Treatment of NOB with a rat liver S-9 mixture led to the formation of 3?-demethyl-NOB, while that of LT did not. We thus suggest that PMFs including NOB have properties distinct from those of general flavonoids for absorption and metabolism in vitro.
Key words: nobiletin; luteolin; metabolism; rat liver S-9 mixture; Caco-2 cells

-31-
Note
Apoptosis-inducing Activity of a Driselase Digest Fraction
of Green Tea Residue

Yuko KATSUNO, Yu KOYAMA, Kouichi SAEKI, Masaki SAZUKA, Katsumasa OOKAWA,＊
and Mamoru ISEMURA・

School of Food and Nutritional Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan
＊Shizuoka Industrial Research Institute of Shizuoka Prefecture, Makigaya, Shizuoka 421-1221, Japan

Received July, 3, 2000; Accepted August 11, 2000
We enzymatically digested green tea residue with Driselase, a crude preparation containing cellulase, pectinase and proteases, in order to examine the potential usefulness of the residue. A fraction of the digest soluble in 70％ ethanol was found to induce the death of U937 human histiocytic lymphoma cells by apoptosis. Other enzyme preparations gave similar products with cell death-inducing activity of varing potency. The green tea residue may therefore be a useful source of potential agents with anti-cancer activity.
Key words: Driselase; pectinase; green tea; apoptosis; anti-cancer activity

-32-
Note
Uptake and Accumulation of Exogenous Docosahexaenoic Acid by Chlorella

Masahiro HAYASHI,1,＊ Tsugiyo YUKINO,1 Isao MARUYAMA,2
Sumiko KIDO,2 and Shozaburo KITAOKA3

1Department of Food Science and Technology, Minami-Kyushu University, Takanabe,
Miyazaki 884-0003, Japan
2Chlorella Industry Co. Ltd., Chikugo, Fukuoka 833-0056, Japan
3Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Osaka 599-8531, Japan

Received July 3, 2000; Accepted August 11, 2000
Tuna oil or its hydrolysate was added to a culture of Chlorella for its nutritional fortification as a feed for rotifer. Exogenous docosahexaenoic acid (DHA) in its free form was taken up by the cells of Chlorella vulgaris strain K-22 and by other strains, but tuna oil was not taken up by the cells. Accumulated DHA was found by electron microscopy in the cells in oil droplets. All strains of Chlorella used in these experiments took up exogenous DHA into the cells. It seems that the structure of the cell wall did not affect the uptake of DHA into the Chlorella cells.
Key words: Chlorella; docosahexaenoic acid; DHA

-33-
Note
Characterization of PBZ1, a Probenazole-inducible Gene,
in Suspension-Cultured Rice Cells.

Hideo NAKASHITA,1 Keiko YOSHIOKA,1§ Masayuki TAKAYAMA,1,2 Rie KUGA,1,3 Naoki MIDOH,4
Ron USAMI,2 Koki HORIKOSHI,2 Katsuyoshi YONEYAMA,3 and Isamu YAMAGUCHI1＊

1RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585, Japan
3Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
4Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku,
Yokohama, Kanagawa 222-0002, Japan

Received July 7, 2000; Accepted September 19, 2000
Probenazole (PBZ) induces non-race specific resistance in rice plants against rice blast fungus and PBZ1 was identified as a PBZ-inducible gene from rice. The induction of PBZ1 expression in suspension-cultured rice cells was investigated. Northern blot analysis indicated that PBZ1 was induced by PBZ in a dose-dependent manner. Enzyme-linked immunosorbent assay (ELISA) showed a dose and time-dependent accumulation of PBZ1 protein. Both mRNA and protein analysis showed that PBZ1 was not induced by salicylic acid or an active metabolite, 1,2-benzisothiazole-1,1-dioxide.
Key words: systemic acquired resistance; SAR; rice; probenazole

-34-
Note
Sequence Analysis and Overexpression of a Pectin Lyase Gene (pel1)
from Aspergillus oryzae KBN616

Noriyuki KITAMOTO,・ Shoko YOSHINO-YASUDA, Kunio OHMIYA,＊ and Norihiro TSUKAGOSHI＊＊

Food Research Institute, Aichi Prefectural Government, 2-1-1 Shinpukuji-cho, Nishi-ku,
Nagoya 451-0083, Japan
＊Faculty of Bioresources, Mie University, Tsu 514-8507, Japan
＊＊Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya 464-8601, Japan

Received July 14, 2000; Accepted September 8, 2000
A gene (pel1) encoding pectin lyase (Pel1) was isolated from a shoyu koji mold, Aspergillus oryzae KBN616, and characterized. The structural gene comprised 1,196 bp with a single intron. The ORF encoded 381 amino acids with a signal peptide of 20 amino acids. The deduced amino acid sequence showed high similarity to those of Aspergillus niger pectin lyases and Glomerella cingulata PnlA. The pel1 gene was successfully overexpressed under the promoter of the A. oryzae TEF1 gene. The molecular mass of the recombinant pectin lyase substantially coincided with that calculated based on nucleotide sequence.
Key words: Aspergillus oryzae; gene cloning; pectin lyase; overexpression

-35-
Note
Effects of the Escherichia coli sfsA Gene on mal Genes Expression
and a DNA Binding Activity of SfsA

Kazuhiko TAKEDA, Chiyuri AKIMOTO, and Makoto KAWAMUKAI＊

Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu, Matsue,
Shimane 690-8504, Japan

Received July 17, 2000; Accepted August 24, 2000
The sfsA gene was identified as one of the sfs genes the over-expression of which stimulates maltose fermentation of the Mal-- Escherichia coli strain MK2001 (crp＊1, cya:Kmr). Expression from the malPQ promoter, which was measured using a chromosomally integrated malPp-lacZ fusion, was induced by over-expressing the sfsA gene in the crp＊1, cya:Kmr strain. The level of the MalE protein was increased in crp＊1, cya:Kmr cells over-producing SfsA. The SfsA protein was purified to homogeneity and tested for DNA binding activity. The purified SfsA protein binds to DNA non-specifically. All these results may suggest that SfsA functions as a DNA binding protein to induce the mal genes in coordination with CRP＊1.
Key words: SfsA; maltose; Escherichia coli

-36-
Note
Primary Culture of Chicken Hepatocytes as an in Vitro Model
for Determining the Influence of Dioxin

De-Xing HOU,1・ Tomohiro KUNITAKE,1 Jun KUSUDA,2 and Makoto FUJII1

1Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, 1-21-24
Korimoto, Kagoshima 890-0065, Japan
2Division of Genetic Resource, National Institute of Health, 1-5 Toyama, Shinjuku-ku, Tokyo 162-0052, Japan

Received July 21, 2000; Accepted August 28, 2000
An easy method for primary culture of chicken hepatocytes was developed to study the influence of dioxin on birds. Chicken hepatocytes could maintain gene expression and protein secretion of albumin for a long period in serum-free medium with free atmosphere exchange at 37°C. Moreover, the cells showed a sensitive response to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) by monitoring the expression of P450 1A, theta GST (θ-GST) and albumin genes.
Key words: chicken hepatocytes; primary culture; TCDD; P450 1A; albumin

-37-
Note
Crystallization and Structural Analysis of Intact Maltotetraose-forming
Exo-amylase from Pseudomonas stutzeri

Yoshihiro MEZAKI, Yoshio KATSUYA, Michio KUBOTA,＊ and Yoshiki MATSUURA＊＊

Hyogo Prefectural Institute of Industrial Research, 3-1-12, Yukihira-chou, Suma-ku, Kobe 654-0037, Japan
＊Hayashibara Biochemical Laboratories Inc., 7-7, Amase-minami, Okayama 700-0834, Japan
＊＊Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan

Received July 21, 2000; Accepted August 21, 2000
The intact maltotetraose-forming exo-amylase from Pseudomonas stutzeri (G4-1), which has a raw starch binding domain, has been crystallized. The structure was identified (PDB entry 1GCY) by the molecular replacement method using the structure of its catalytic domain (G4-2). The result showed that the raw starch binding domain is in a disordered state, the corresponding electron densities being almost invisible. Superposition of these two enzyme forms showed evidence for the possible location of the raw starch binding domain (SBD). This crystal is a novel case, in that it forms a regular lattice incorporating flexibly bound SBD in the channel of crystal packing of the catalytic domains.
Key words: exo-amylase; raw starch binding domain; X-ray analysis; domain structure; disorder

-38-
Note
A Cryptic Plasmid, pAO1, from a Compost Bacterium, Bacillus sp.

Shuichi KARITA,・・ Akihito OHTAKI,＊ Mina NOBORIKAWA, and Kiyohiko NAKASAKI＊

Center for Molecular Biology and Genetics, Mie University, Tsu 514-8507, Japan
＊Department of Materials Science and Chemical Engineering, Faculty of Engineering, Shizuoka University,
Hamamatsu 432-8561, Japan

Received July 26, 2000; Accepted September 12, 2000
The complete nucleotide sequence of a new cryptic plasmid, pAO1 isolated from a compost bacterium Bacillus sp., has been analyzed. Analysis of the PCR-based 16S rRNA sequence showed the bacterium harboring pAO1 was closely related to Bacillus pallidus. The plasmid pAO1 was 3,325 bp in size. Two open reading frames, ORF1 and ORF2, encoding putative polypeptides of 248 and 290 amino acids, respectively, were identified within the sequence. The ORF1 has a limited sequence similarity to an integrase/recombinase, while the ORF2 has high similarity with the replication protein of pBC1 from Bacillus coagulans. A putative origin sequence for a plus-strand was located between ORFs. Southern blot analysis indicates this plasmid replicates via a rolling circle-type mechanism.
Key words: cryptic plasmid; compost bacterium; Bacillus pallidus

-39-
Note
Translational Regulation of the mRNA Encoding the Eukaryotic Translation
Initiation Factor 4E in Xenopus

Motoaki WAKIYAMA, Motoki SAIGOH, Kayoko IKEDA, Aki SUZUKI, and Kin-ichiro MIURA

Institute for Biomolecular Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan

Received August 1, 2000; Accepted August 30, 2000
We have cloned the cDNA for Xenopus eukaryotic translation initiation factor 4E (eIF4E). Here we show that translation of a luciferase mRNA that contains the 5? untranslated region derived from Xenopus eIF4E is active in fertilized eggs, but is repressed in oocytes. The results suggest that the expression of Xenopus eIF4E is regulated at the translation level.
Key words: translational regulation; translation initiation factor 4E; Xenopus; oocytes

-40-
Note
Analysis of the Isoform of Xenopus Euakryotic Translation Initiation Factor 4E

Motoaki WAKIYAMA, Aki SUZUKI, Motoki SAIGOH, Nobuya SAKAI, Hiroshi MIYOSHI,
Shuichi KOJIMA, and Kin-ichiro MIURA

Institute for Biomolecular Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan

Received August 1, 2000; Accepted September 5, 2000
We have found two isoforms of the eukaryotic translation initiation factor 4E (eIF4E) in Xenopus laevis. These proteins differ in length by 18 amino acids. Overexpression of either of the two eIF4E proteins modestly increase translation in Xenopus oocytes. The results suggest that both of these two isoforms function in translation.
Key words: translation initiation factor 4E; isoform; Xenopus

-41-
Preliminary Communication
Enterostatin (VPDPR) Has Anti-analgesic and Anti-amnesic Activities

Yasuyuki TAKENAKA,1 Futoshi NAKAMURA,1 YUNDEN Jinsmaa,1 Andrzej W. LIPKOWSKI,1,2
and Masaaki YOSHIKAWA1

1Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan
2Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

Received August 11, 2000; Accepted October 11, 2000
Enterostatin (VPDPR), an anorexigenic peptide derived from the amino terminus of procolipase, significantly inhibited analgesia induced by the μ-opioid agonist morphine (5 mg/kg, s.c.) after i.c.v. administration to mice at a dose of 100 nmol. On the other hand, VPDPR (~200 nmol, i.c.v.) did not attenuate analgesia induced by the κ-opioid agonist D-Phe-D-Phe-D-Nle-D-Arg-NH2 (100 μg/mouse, i.c.v.) or δ-opioid agonist DTLET (4 nmol/mouse, i.c.v.). VPDPR (100 nmol, i.c.v.) significantly improved amnesia induced by scopolamine (0.2 mg/kg, i.p.) in mice. However, VPDPR did not enhance memory in normal mice at the same dose.
Key words: enterostatin; anti-analgesia; anti-amnesia; anti-opioid; μ-receptor